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Purpose: Mutations of the CYP1B1 gene cause primary congenital glaucoma (PCG), Peters anomaly, and juvenile open-angle glaucoma (JOAG). The aim of this study was to determine the spectrum and role of the CYP1B1 gene in Japanese patients with PCG or JOAG., Methods: Genomic DNA was extracted from the leukocytes of 18 unrelated patients with PCG and 21 unrelated patients with JOAG. All of the patients developed high intraocular pressure (IOP) before the age of 35 years. One hundred unrelated healthy adults with normal IOP were examined in the same way. The three exons of the CYP1B1 gene were amplified by polymerase chain reaction and directly sequenced., Results: Mutational screening and sequence analyses of the CYP1B1 gene revealed four mutations in four patients with PCG: p.Asp192Val, c.4776insAT, p.Val364Met, and p.Asp430Glu. The first three mutations have been reported in other Japanese PCG patients, but Asp430Glu is a new mutation. No mutations were found in the CYP1B1 gene of the JOAG patients., Conclusions: PCG in approximately 20% of Japanese patients may be associated with CYP1B1 mutations, but JOAG is not. The three mutations p.Asp192Val, c.4776insAT, and p.Val364Met appear to be common in the Japanese population and might be useful in genetic screening for PCG.

The etiology of recurrent pregnancy loss (RPL) remains unclear, but it may be related to a possible genetic predisposition together with involvement of environmental factors. We examined the relation between RPL and polymorphisms in four genes, human aryl hydrocarbon (Ah) receptor, cytochrome P450 (CYP) 1A1, CYP1A2 and CYP1B1, which are involved in the metabolism of a wide range of environmental toxins and carcinogens. All cases and controls were women resident in Sapporo, Japan and the surrounding area. The Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotypes were assessed in 113 Japanese women with recurrent pregnancy loss (RPL) and 203 ethnically matched women experiencing at least one live birth and no spontaneous abortion (control). No significant differences in Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotype frequencies were found between the women with RPL and the controls [Ah receptor: Arg/Arg (reference); Arg/Lys and Lys/Lys, odds ratio (OR)=0.67; 95% confidence interval (CI)=0.40-1.11, CYP1A1: m1m1 (reference); m1m2 and m2m2, OR = 0.86; 95% CI = 0.53-1.40, CYP1A2: C/C and C/A (reference); A/A, OR = 1.16; 95% CI = 0.71-1.88, CYP1B1: Leu/Leu (reference); Leu/Val and Val/Val, OR = 1.18; 95% CI = 0.68-2.02]. The present study suggests that the Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not major genetic regulators in RPL.

We developed genotyping assays for CYP2A6*7 (Ile471Thr) and CYP2A6*8 (Arg485Leu). We found higher allelic frequencies in Japanese and Chinese versus Caucasians and identified an allele in which both substitutions occur together (CYP2A6*10). We created a homology model for predicting the impact of allelic variants on enzymatic activity and subsequently tested this in vivo in a pilot kinetic study. Consistent with our homology model predictions, we found (i) that CYP2A6*7 produces an enzyme that has decreased (not inactive) activity for metabolizing nicotine and coumarin; (ii) that CYP2A6*8 is unlikely to affect catalytic activity in vivo; and (iii) that having both substitutions together on an allele (CYP2A6*10) dramatically reduces function and may be fully inactive for some substrates. In conclusion, this study identifies, at relatively high frequency in Asians, an allele with decreased activity (may be substrate selective), a fully functional allele, and an allele containing both substitutions in which function is dramatically reduced., ((c)2002 Elsevier Science.)

We previously reported that a comparative pharmacokinetic study with each PPI was designed as an open, randomized, and crossover study of 18 Japanese healthy volunteers who were classified into the homozygous, heterozygous extensive metabolizer and the poor metabolizer based on the CYP2C19 genotype. With at least 1 week washout period between treatments. Plasma concentrations of PPIs and their metabolites were monitored until 12 h after medication. Pharmacokinetic profiles of omeprazole and lansoprazole were well correlated with the CYP2C19 genotype. The heterozygous extensive metabolizer was slightly different from the homozygote, but there was no statistically significant difference. The CYP2C19 genotype dependence found for lansoprazole was not obvious compared with omeprazole. As for rabeprazole, the pharmacokinetic profile was independent of the CYP2C19 genotype. CYP2C19 genotyping can provide a new strategy to choose an optimal regimen, and this genotyping is especially useful for Japanese, as the frequency of poor metabolizers is five times greater than that found among Caucasians.

Background: Omeprazole is mainly metabolized by cytochrome P450 2C19 (CYP2C19) in the liver. Rabeprazole, on the other hand, is mainly metabolized to thioether-rabeprazole via a non-enzymatic pathway and partially metabolized to demethylated-rabeprazole by CYP2C19 in liver CYP2C19 status may affect cure rate for Helicobacter pylori infection with proton pump inhibitor triple therapy., Aim: To investigate whether genetic polymorphism of CYP2C19 and selected proton pump inhibitors (omeprazole or rabeprazole) were associated with cure rate for Helicobacter pylori infection using triple therapy with omeprazole or rabeprazole, amoxicillin, and clarithromycin., Methods: A total of 170 Helicobacter pylori-positive patients with chronic gastritis were randomized to receive one of the following Helicobacter pylori eradication regimens; OAC (omeprazole 20 mg bd, amoxycillin 750 mg bd and clarithromycin 400 mg bd for 1 week) and RAC (rabeprazole 20 mg bd, amoxycillin 750 mg bd and clarithromycin 400 mg bd for 1 week). The CYP2C19 genotype; wild-type or two mutant genes (ml in exon 5 and m2 in exon 4), or both, were identified by polymerase chain reaction-restriction fragment length polymorphism., Results: In DAC regimen, cure rate (per protocol analysis) was 73.3% in homozygous extensive metabolizers, 86.1% in heterozygous extensive metabolizers, and 85.0% in poor metabolizers. In RAC regimen, the cure rate was 81.0% in homozygous extensive metabolizers, 82.9% in heterozygous extensive metabolizers, and 87.5% in poor metabolizers. Cure rate was not significantly different between the CYP2C19 genotypes in both regimens., Conclusion: Triple therapy with proton pump inhibitor (omeprazole or rabeprazole), amoxycillin, and clarithromycin is sufficiently effective in cure of Helicobacter pylori infection regardless of CYP2C19 status.

This study determined the genotypes of the CYP2A6 gene in 96 smokers and 141 non-smokers in a Japanese population. The frequencies of wild-type of the CYP2A6* 1 and those with a whole deletion of the CYP2A6 gene were 93 (96.9%) and 3 (3.1%) in 96 smokers, and 134 (95.0%) and 7 (5.0%) in non-smokers, respectively. In addition, neither the CYP2A6* 2 nor CYP2A6* 3 alleles were observed in the population studied. There were no significant differences in the CYP2A6 genotype frequencies between smokers and non-smokers, as well as in the number of cigarettes smoked and the nicotine amounts inhaled per day between the CYP2A6* 1 and the deletion of CYP2A6. These results suggest that either the deletion or non-deletion of the CYP2A6 gene shows no significant effect on smoking behavior for the Japanese population examined.

Purpose: To investigate CYP1B1 gene mutations in Japanese patients with primary congenital glaucoma (PCG)., Methods: Sixty-five unrelated Japanese patients with PCG were screened by PCR-single-strand conformational polymorphism (SSCP) analysis followed by direct sequencing. No patients were offspring of consanguineous marriages, a common occurrence among patients in previous reports. PCG haplotypes were constructed with intragenic polymorphisms in affected individuals. Three-dimensional atomic structures of human CYP1B1 and four mutant CYP1B1 sequences representing missense mutations were assembled using homology modeling and were regularized by an energy-minimization procedure., Results: Eleven novel mutations, including seven definite and four probable mutations, were detected in 13 (20%) of the 65 unrelated patients. Of the seven definite mutations, three were predicted to truncate the CYP1B1 open reading frame. The other four were missense mutations (Asp192Val, Ala330Phe, Val364Met, and Arg444Gln), all located in conserved core structures determining proper folding and heme-binding ability of cytochrome P450 molecules. Molecular modeling demonstrated that two of four mutations in positions 330 and 364 were structurally neutral, but Arg444Gln caused significant structural change. Of the four probable mutations, three were missense (Val198Ile, Val320Leu, and Glu499Gly); the other was a base substitution in the noncoding region of exon 1., Conclusions: The 11 varied CYP1B1 mutations found in 13 unrelated Japanese patients with sporadic occurrence of PCG represent an allelic heterogeneity and may be unique to a specific population.

Objective: To investigate putative differences in CYP3A activity between European American and Japanese subjects using midazolam as an in vivo probe., Methods: Midazolam was administered orally (2 mg) to 22 young healthy Japanese men and, on a separate occasion, to 19 of these by the intravenous route (1 mg). The disposition of the drug and its 1'-hydroxy metabolite were determined and compared with data collected in a similar fashion in 20 young healthy European American men., Results: Plasma concentrations of midazolam, especially those attained soon after drug administration, were higher after intravenous injection in Japanese subjects than those in European American men. This observation was associated with smaller initial (2.5-fold) and steady-state (1.8-fold) volumes of distribution for the drug; normalization for body weight only modestly reduced these differences. The systemic clearance value of midazolam was 25% lower (P < .03) in Japanese subjects, but this difference was not apparent after accounting for the smaller body weights of that group. No statistical differences were noted in the elimination half-life (t 1/2) of midazolam between European American and Japanese subjects. Much greater interindividual variability was observed after oral administration compared with intravenous administration, but significant differences were not found between the 2 groups with respect to the maximum midazolam plasma level or its oral clearance. Absolute oral bioavailability and its associated gastrointestinal and hepatic extraction ratios also showed no statistically significant interracial differences., Conclusions: On average, hepatic CYP3A, as measured by the metabolism of midazolam, is lower in young healthy Japanese men compared with similar European Americans. However, there is considerable interindividual variability, and body size appears to be an important determinant. After oral administration, even greater variability in the plasma level-time profile of midazolam is present, and no statistically significant or clinically important interracial/ethnic difference is present. Possibly because of smaller body mass and differences in body composition, midazolam has a smaller distribution volume(s) in Japanese men than in European American men that might be an important factor when drugs are administered intravenously.

A single nucleotide polymorphism (SNP) resulting in a substitution from Gln to His was found in exon 4 of the CYP2B6 gene in Japanese. The frequency of the variant allele was found to be 19.9%. The mutant- and the wild-type enzymes were expressed in Escherichia coli, and the effects of the single amino acid substitution on the catalytic activity were examined by investigating the kinetic profiles of 7-ethoxycoumarin O-deethylase activity. The wild-type enzyme showed typical Michaelis-Menten kinetics, while the mutant-type enzyme represented the sigmoidal kinetics with a higher V(max) value compared to that of the wild-type enzyme. Eadie-Hofstee plots further revealed an existence of allosteric effects for the reaction catalyzed by the variant. This is the first evidence demonstrating that only one amino acid substitution, Gln172His, caused by natural SNP enhances the catalytic activity of CYP by obtaining the character of homotropic cooperativity., (Copyright 2001 Academic Press.)

Purpose: To report a novel missense mutation and DNA polymorphism of the CYP1B1gene in Japanese patients with primary congenital glaucoma., Methods: A series of 11 unrelated patients with primary congenital glaucoma was examined. Patients were followed in the Kagoshima University Hospital between 1979 and 1998. DNA was extracted from leukocytes of the patients, their families, and unrelated healthy individuals. Amplicons spanning the coding regions of the CYP1B1 gene were examined by direct sequencing and enzyme-restriction detection., Results: In the 11 unrelated patients, besides the previously reported insertional mutation (1620 ins G), a novel missense mutation was identified at codons 444 to replace arginine with glutamine (R444Q) in one patient. The novel missense mutation cosegregated in the relevant family as an autosomal recessive pattern and was not found in other patients or control individuals. In addition, five polymorphic sites were found at codons 48, 119, 330, 432, and 449. These polymorphic alleles did not cosegregate with the disease, and they were found in healthy individuals as well., Conclusions: Approximately 20% of Japanese patients with primary congenital glaucoma may be affected by mutations in the CYP1B1 gene. Further studies are justified to explore whether a relationship exists between the phenotypic expressivity of the disease and the type of mutation.

During the course of investigating the frequency of a CYP2A6 whole deletion-type polymorphism (CYP2A6*4C) in Japanese, an unexpectedly large population of heterozygotes for CYP2A6*4C and the wild-type (CYP2A6*1A) was found. Cloning of a cDNA encoding CYP2A6 from the liver of individuals judged as heterozygotes for CYP2A6*4C and the CYP2A6*1A was carried out to identify the causal allele(s) responsible for a possible overestimation. A clone isolated from the liver cDNA library possessed 58 bp sequences in the 3'-untranslated region, which was replaced with the corresponding region of the CYP2A7 gene. The same gene conversion existed in the genomic DNA, indicating that the replacement was not a cloning artifact. Based on the gene structure of the allele (CYP2A6*1B), this variant was thought to be one of the causal alleles responsible for overestimation of heterozygotes for CYP2A6*4C and CYP2A6* A. To investigate this further, we developed a genotyping method which could distinguish the CYP2A6*A, CYP2A6*1B and CYP2A6*4C alleles from each other. The results clearly showed that CYP2A6*1B was the sole allele responsible for the overestimation. We conclude that the new genotyping method allows determination of six genotypes of the CYP2A6 gene, simultaneously and precisely, in both Oriental and Caucasian populations.

Objective: To clarify developmental changes in the pharmacokinetics and dynamics of warfarin enantiomers to establish rational pediatric dosage., Methods: Plasma concentrations of unbound warfarin enantiomers, vitamin K1 and vitamin K-dependent proteins (that is, prothrombin fragments 1+2, protein C, and the protein-induced by vitamin K absence) and international normalized ratio were measured in 38 prepubertal (1 to 11 years), 15 pubertal (12 to 18 years), and 81 adult (37 to 76 years) patients given long-term warfarin therapy. Unbound oral clearance values for warfarin enantiomers and its body weight-, body surface area-, and liver weight-normalized values, as well as the pharmacodynamic parameters, were compared among the groups., Results: The prepubertal, pubertal, and adult patients exhibited comparable mean plasma concentrations of unbound warfarin enantiomers for pharmacologically more active (S)-warfarin. Although the unbound oral clearance of (S)-warfarin for the prepubertal patients was significantly (P < .01) less than that for the adult group (346 versus 637 mL/min), the body weight-normalized unbound oral clearance for the prepubertal patients was significantly (P < .01) greater than that for the adults and showed a negative correlation (P < .05) with age. In contrast, no differences were observed in the liver weight-normalized unbound oral clearance for (S)-warfarin between the prepubertal and adult groups. The prepubertal patients showed significantly (P < .01 or .05) lower plasma concentrations of protein C and prothrombin fragments 1+2 and greater international normalized ratio and international normalized ratio/dose than the adults. In contrast, the pubertal patients showed largely similar pharmacokinetic and pharmacodynamic properties to adults., Conclusion: Liver weight may be a better parameter than body weight for estimating the warfarin doses for prepubertal patients on the basis of the corresponding adult values. Augmented responses to warfarin in children should also be taken into account for estimating warfarin doses for children.

1. Race-related differences in the frequency distribution of genetic polymorphisms in the CYP1A1 and CYP1B1 genes were studied in 39 Japanese and 45 Caucasians. 2. Four types of CYP1A1 polymorphism, namely m1 (a nucleotide change at T6235C in the 3'-flanking region), m2 (A4889G at exon 7), m3 (T5639C in the 3'-flanking region) and m4 (C4887A at exon 7), and three types of CYP1B1 genetic polymorphism, namely m1 (C488G and G701T leading to Arg48Gly and Ala119Ser exchanges respectively), m2 (C1294G leading to a Leu432Val exchange) and m3 (A1358G leading to an Asn453Ser exchange) were studied. 3. The distribution of the m1-, m2-, m3-, and m4-types of CYP1A1 polymorphism in the Japanese population was 30.8, 17.9, 0 and 0% respectively; those in Caucasians were 3.3, 6.7, 0 and 2.2% respectively. Two types (m1, and m2) of CYP1B1 polymorphism were expressed at 14.1 and 21.8% respectively in the Japanese, and by 28.9 and 37.5% respectively in the Caucasian. Ethnic differences were also noted in the m3-type CYP1B1 polymorphism in which the incidence in Caucasians was 23.9%, whereas no cases in the 39 Japanese subjects were observed. 4. No apparent association was found in the incidence in each of the genetic polymorphisms of CYP1A1 and CYP1B1 genes, nor in methylenetetrahydrofolate reductase gene, except that the occurrence of the m2-type of CYP1A1 genetic polymorphism was related to that of the m1-type CYP1A1 polymorphism in the Japanese population. 5. These results suggest that there are race-related differences in the occurrence of genetic polymorphisms in both CYP1A1 and CYP1B1 genes in Japanese and Caucasian populations and that these differences in P450 genetic polymorphisms may, in part, cause differences in the occurrence of lung and breast cancers in these ethnic groups.

Cytochrome P450 1B1 (CYP1B1) participates in the metabolic activation of a number of procarcinogens including benzo[a]pyrene and the hydroxylation of 17beta-estradiol at the C-4 position. In this study, we investigated the association between CYP1B1 genetic polymorphism and breast or lung cancer incidence. The Ala-Ser polymorphism at codon 119 in presumed substrate recognition site 1 was significantly associated with the incidence of breast or squamous cell carcinoma of the lung. On the other hand, Leu-Val polymorphism at codon 432 did not show any association to the cancers. An allele containing both Ala and Leu simultaneously, comprised 75% of alleles among 315 Japanese healthy controls, was significantly inversely associated with breast cancer incidence. When expressed in a recombinant system, this CYP1B1 cDNA showed the lowest 17beta-estradiol 4-hydroxylase activity among four different variant forms of CYP1B1. Thus, inter-individual differences in activation of procarcinogens or metabolism of oestrogen originating from genetic polymorphisms of the human CYP1B1 gene may contribute to the susceptibility of human cancers.

Genotypes of CYP2A6, namely CYP2A6(*)1 (wild-type), CYP2A6(*)2, and CYP2A6(*)3, were examined in liver DNA of 39 Japanese and 43 Caucasians using two-step polymerase chain reaction (PCR) methods. We first amplified a DNA fragment (1725 bp) located between near middle of exon 1 and end of exon 4 of the CYP2A6 gene and further amplified using a forward primer 't' or 'mut' (middle of exon 3) and a reverse primer 'E3R' (middle of intron 3) for the detection of CYP2A6(*)2-genetic polymorphism. The 1725 bp fragment was also used for the amplification between exon 3 and near middle of intron 3 of the CYP2A6 gene and the fragment thus obtained digested with XcmI or DdeI to detect and confirm the CYP2A6(*)2- and CYP2A6(*)3-types, respectively. Only one DNA sample from a Japanese origin (J18) was not amplified by CYP2A6-specific primers; liver microsomes from this individual had very low activity of coumarin 7-hydroxylation and were devoid of protein(s) immunoreactive to anti-CYP2A6 antibody. Thus, this individual was suggested to be due to the gene deletion in CYP2A6. By analyzing the remaining 38 Japanese and 43 Caucasians, we found that there were no cases of CYP2A6(*)3-type polymorphism in the samples examined in this study, and no cases of CYP2A6(*)2-type polymorphism in the Japanese samples. Of Caucasians studied two individuals were classified into heterozygous CYP2A6(*)1/(*)2-type. Liver microsomal coumarin 7-hydroxylation activities in these two Caucasians were found to be lower than those of the other 41 Caucasians. Kinetic analysis showed that two CYP2A6(*)1/(*)2 individuals had a very low ratio of V(max) to K(m) for nicotine C-oxidation as well as coumarin 7-hydroxylation in liver microsomes, compared with those of homozygous CYP2A6(*)1-type. These results suggest that among 39 Japanese and 43 Caucasians examined one Japanese is classified to be CYP2A6 gene deletion and two Caucasians are heterozygous CYP2A6(*)1/(*)2-genotype. Thus the race-related differences in the occurrence of CYP2A6 genetic polymorphisms were supported.

CYP2C9 catalyzes the metabolism of important drugs such as phenytoin, S-warfarin, tolbutamide, losartan, and nonsteroidal anti-inflammatory drugs. A functional polymorphism of the CYP2C9 gene has been described. The single-base mutation of A1061C (Ile359Leu) in the CYP2C9 gene termed CYP2C9*3 was found at a frequency of about 2.1% in Japanese. We developed a rapid mutation analysis method for detecting the CYP2C9*1 genotype. This method is a marriage of two emerging technologies: allele-specific amplification primers for target DNA and a new double-stranded DNA-selective fluorescent dye, SYBR Green. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. We applied this procedure to DNA extracted from the blood of healthy Japanese volunteers. The CYP2C9 wild-type CYP2C9*1/CYP2C9*1 and heterozygous CYP2C9*1/CYP2C9*3 genotypes of the CYP2C9 alleles detected by the assay were consistent with the results obtained from restriction enzyme cleavage. No genotype of CYP2C9*3/CYP2C9*3 was found in these samples. Using plasmid DNA containing a point mutation of CYP2C9*3 as template, the assay separated the three genotypes. We conclude that this simple, rapid, and inexpensive procedure is applicable to routine high-throughput assays., (Copyright 1999 Academic Press.)

Regio- and stereoselective hydroxylation of phenytoin was determined in liver microsomes of nine extensive (EM) and three poor metabolizers (PM) of mephenytoin. Hydroxyphenytoins (HPPH) were isolated and quantified after separation into four regio- and stereoisomers. The total rates of microsomal phenytoin 4'- hydroxylation were approximately 3-fold higher than those of 3'-hydroxylation, and not significantly different in EM and PM. Formation of 4'-(R)-HPPH was 4.4-fold higher in EM than in PM, whereas no clear differences between EM and PM were detected in the formation of 4'-(S)-, 3'-(R)-, and 3'-(S)-HPPH. Cytochrome P450 (CYP)2C9, expressed in a fission yeast, Schizosaccharomyces pombe, catalyzed the formation of 4'-(R)- and 4'-(S)-HPPH stereoselectively, as observed with EM, in which predominantly 4'-(S)-HPPH was formed. Recombinant CYP2C19 was more stereoselective for 4'-(R)-HPPH formation. These results, in addition to inhibition experiments with anti-human CYP2C antibody, indicate that phenytoin hydroxylation is mainly catalyzed by CYP2C9. Furthermore, CYP2C19 showed limited contribution to phenytoin 4'-hydroxylation with a different chiral preference from CYP2C9.

Objective: Omeprazole is metabolized by genetically determined S-mephenytoin 4'-hydroxylase (CYP2C19) in the liver. This study aimed to determine whether the effect of omeprazole on intragastric pH depends on CYP2C19 genotype status., Methods: CYP2C19 genotype status for 2 mutations associated with the poor metabolizer phenotype was determined by a polymerase chain reaction-restriction fragment length polymorphism method in 16 healthy volunteers. Helicobacterpylori status was determined by serology and the [13C]urea breath test. After a single oral administration of 20 mg omeprazole or a placebo, intragastric pH values were recorded for 24 hours. Plasma levels of omeprazole and its 2 metabolites and gastrin were measured before and 1, 2, 3, 5, 7, 10, and 24 hours after administration., Results: Fifteen of the 16 subjects were H pylori negative. Five of the 15 subjects were homozygous extensive metabolizers, 4 were heterozygous extensive metabolizers, and 6 were poor metabolizers. After omeprazole administration, significant differences in mean intragastric pH values and plasma levels of gastrin, omeprazole and its metabolites were observed among the 3 groups, whereas no significant differences in these parameters were observed with the placebo administration., Conclusions: The effect of omeprazole on intragastric pH significantly depends on CYP2C19 genotype status. The genotyping test of CYP2C19 may be useful for an optimal prescription of omeprazole.

Ethnic differences in the pharmacokinetics of nifedipine, a substrate of CYP3A, and in CYP3A7 expression have been reported. The aim of the present study was to measure the protein levels of CYP3A4, CYP3A5, and CYP3A7 and nifedipine oxidation activity in hepatic microsomes from 15 Caucasian and 15 Japanese patients for comparison between the two ethnic groups. Nifedipine oxidation activity and CYP3A4 protein level were well correlated. No significant difference between Caucasian and Japanese microsomal samples was found in nifedipine oxidation activity or in the CYP3A4 protein level. CYP3A5 was detected in 6 of 15 Caucasian samples and in 5 of 15 Japanese samples, but no ethnic difference was found in either the frequency of expression or its protein level. CYP3A7 was found in 10 of 15 Caucasian samples and in 14 of 15 Japanese samples. Although the estimated CYP3A7 protein level was higher in the Japanese than in the Caucasian samples, its protein level was much lower than that of CYP3A4. These results imply that the contribution of CYP3A5 or CYP3A7 to the purported Caucasian-Japanese ethnic difference in the overall CYP3A activity seems to be small.

Omeprazole is widely used for the treatment of Helicobacter pylori infection. It is metabolized by cytochrome P450 2C19 enzyme (CYP2C19) in the liver. Because this enzyme exhibits a genetic polymorphism, patients with low metabolic activity (poor metabolizers) may be exposed to higher concentrations of this drug than are patients who are extensive metabolizers. Eighty-six patients with cultured H. pylori-positive gastritis or peptic ulcers who completed the treatment and assessment of anti-H. pylori therapy were analyzed for CYP2C19 genotyping using a polymerase chain reaction-restriction fragment length polymorphism method [the wild-type or two mutant genes (ml in exon 5 and m2 in exon 4), or both]. Patients were classified into three groups according to the H. pylori eradication regimen: group I (n = 21; omeprazole 40mg/ day and amoxicillin 2000mg/day for 1 week); group II (n = 21; group I regimen plus sucralfate 4000mg/day, for 1 week); group III (n = 44; group I regimen plus clarithromycin 800mg/day, for 1 week). The combination of two mutant alleles (ml/ml, ml/m2, m2/m2-poor metabolizers) was observed in 13 of 86 patients (15%), and all poor metabolizer patients achieved H. pylori eradication regardless of their treatment regimens. In addition, the eradication rates of the poor metabolizers were significantly higher than those of other genotypes who carry homozygous or heterozygous normal allele (extensive metabolizers) in group I or groups I and II combined. CYP2C19 genotyping can provide a new strategy to choose an optimal regimen, and this genotyping is especially useful for Japanese, as the frequency of poor metabolizers is five times greater than that found among Caucasians.

Interindividual variation of drug effects in humans can be attributed to many factors. Among the factors, the rate of drug metabolism has been regarded as the most important one. A genetic defect of enzymes involved in drug metabolism, particularly cytochrome P450 (CYP), has been believed to be one of the important causal factors of adverse drug reactions. There are multiple gene mutations for CYP causing the poor metabolizer phenotype. The occurrence of genetic polymorphism has been seen in genes for CYP1A1, CYP2A6, CYP2C9, CYP2D19, CYP2D6, CYP2E1 and CYP3A5. Among them, the molecular mechanisms of genetic polymorphisms of CYP2D6 (debrisoquine/sparteine type) and CYP2A6 (coumarin type) in Japanese have been the focus of investigations. Only 20-30% of the Japanese population that shows the CYP2D6 poor metabolizer phenotype can be diagnosed by gene analysis. By other means, we found two new mutations, CYP2D6/J9 and CYP2D6/C8, in Japanese. With regard to CYP2A6, we discovered SM-12502, a new probe drug in addition to coumarin, that is currently under development; this drug is mainly metabolized by CYP2A6. Using this drug as a probe, we found poor metabolizers and analyzed the genes for CYP2A6. We found a new mutation (CYP2A6 whole deletion) responsible for the poor metabolizer phenotype. These results should contribute to the selection of an effective drug prescription and a reduced incidence of adverse effects.

The coincidence of mutated alleles of CYP2C18 and CYP2C19 was studied in 154 Japanese subjects. The mutant alleles of CYP2C18 studied were CYP2C18m1 (T204 --> A substitution in exon 2) and CYP2C18mFR (A-460 --> T substitution in the 5'-flanking region), and those of CYP2C19 were CYP2C19m1 (G689 --> A substitution in exon 5) and CYP2C19m2 (G636 --> A substitution in exon 4). They were identified by polymerase chain reaction and restriction fragment length polymorphism. The results indicate that genotypes of CYP2C18m1 and CYP2C18mFR are completely coincident with those of CYP2C19m2 and CYP2C19m1, respectively. The finding suggests that the mutations of CYP2C18 and CYP2C19 examined in the present study are very closely linked with each other (i.e. CYP2C18m1 vs CYP2C19m2 and CYP2C18mFR vs CYP2C19m1), at least in a Japanese population.

Genotypings of two mutations (*2 and *3) in CYP2C19 and the amino acid variants (Arg144/Cys, Tyr358/Cys, Ile359/Leu, and Gly417/Asp) in CYP2C9 were carried out in 140 unrelated Japanese subjects. Thirty-three subjects (23.6%) were genotypically identified as poor metabolizers of CYP2C19, and the allele frequencies of the CYP2C19*2 and CYP2C19*3 were 0.35 and 0.11, respectively. The authors' findings are in agreement with the 18% to 23% prevalence of poor metabolizers in the Japanese populations previously phenotyped. In CYP2C9, all subjects were homozygous (CYP2C9*1) for Arg144, Tyr358, Ile359, and Gly417, except for five subjects (3.6%) who were heterozygous for the Leu359 (CYP2C9*3). The frequencies of Arg144, Tyr358, Ile359, Leu359, and Gly417 variants were 1.0, 1.0, 0.982, 0.018, and 1.0, respectively. The low frequency of the Cys144 allele (CYP2C9*2) in the Japanese population is different from the frequency recently found in British subjects (allele frequency, 0.125 to 0.192). The results suggest that the known interindividual variations in the CYP2C9 sequence among Japanese subjects is small, and that Ile359/Leu is one possible site showing interracial polymorphism.

Genetic polymorphism of drug metabolizing enzymes, particularly cytochrome P450(CYP), is an important cause of adverse drug reactions. Multiple gene mutations in CYP have been shown to be phenotype. The occurrence of genetic polymorphism has been seen in genes for CYP1A1, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A5. This review discusses the molecular mechanism of two genetic polymorphisms, debrisoquine/sparteine (CYP2D6) coumarin (CYP2A6) polymorphisms. In addition, elucidation of gene mutations of CYP2D6 and CYP2A6 in Japanese will be discussed.

1. Two different types of genetic polymorphisms in each of CYP2C18 and CYP2C19 genes were examined and compared with respect to their frequencies in distribution in liver DNA of 39 Japanese and 45 Caucasians. 2. Individuals who were classified into CYP2C19m1 (as detected with SmaI digestion) in exon 5 of CYP2C19 gene were found to display a CYP2C18m1 polymorphism (as detected with DdeI digestion) in the 5'-flanking region of CYP2C18 gene in Japanese and Caucasian populations. The Japanese subjects who were classified into CYP2C19m2 (as detected with BamHI digestion) in exon 4 of CYP2C19 gene were found to have a CYP2C18m2 genetic polymorphism (as detected with Tsp509I digestion) in exon 2 of CYP2C18 gene. None of the Caucasians had the CYP2C18m2 nor CYP2C19m2 alleles. 3. Frequencies in two types (C416T in exon 3, A1061C in exon 7) of CYP2C9 genetic polymorphism were found to be independent to those of CYP2C18 and CYP2C19 genetic polymorphisms in these samples. 4. Thus, the results suggest that the CYP2C18 gene is localized very closely to the CYP2C19 gene on the same human chromosome.

1. Tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities were determined in liver microsomes of 39 Japanese and 45 Caucasians genotyped for the cytochrome P450 (P450 or CYP) 2C9 gene into three groups, namely the wild-type (Arg144.Ile359), and two heterozygous Cys allele (Cys144.Ile359) and Leu allele (Arg144.Leu359) variants. 2. Good correlations were found between tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities in liver microsomes of Japanese and Caucasians. Humans with the Cys allele CYP2C9 variant, which was detected in 22% of Caucasians, were found to have similar catalytic rates to those of the wild-type in the oxidations of tolbutamide and racemic warfarin, whereas humans with the Leu allele, which was detected in 8% Japanese and 7% Caucasian samples, had lower catalytic rates than those of other two groups. 3. The rates of 6- and 7-hydroxylation of racemic warfarin were correlated well with those of S-warfarin, but not R-warfarin, in human liver microsomes. 4. Both human liver microsomes and recombinant CYP2C9 catalysed 7-hydroxylation of S-warfarin more extensively than those of R-warfarin. K(m)'s for the 7-hydroxylation of S-warfarin were not very different in liver microsomes of humans with these three genotypes. Anti-CYP2C9 antibodies and sulphaphenazole inhibited the 6- and 7-hydroxylation of S-warfarin, but not R-warfarin, by > 90% and the methyl hydroxylation of tolbutamide by about 50%. 5. These results suggest that humans with Leu allele of CYP2C9 have lower Vmax's for S-warfarin 7-hydroxylation and tolbutamide methyl hydroxylation than those with wild-type and Cys allele CYP2C9, although the K(m)'s are not very different in liver microsomes of these three groups of humans. R-warfarin hydroxylation may be catalysed by P450 enzymes other than CYP2C9 in man.

Objective: To assess the genotype pattern of S-mephenytoin 4'-hydroxylation in an extended Japanese population., Methods: One hundred eighty-six unrelated, healthy Japanese subjects were genotyped for S-mephenytoin 4'-hydroxylase (CYP2C19) according to a genotyping technique to identify the wild-type (wt) gene and two mutations, CYP2C19m1 in exon 5 and CYP2C19m2 in exon 4. Fourty-six of the 186 subjects genotyped were phenotyped with racemic mephenytoin using the conventional 8-hour urine analysis of 4'-hydroxymephenytoin., Results: The frequency of poor metabolizers by the genotyping analysis was 18.8% (35 of the 186 subjects), consisting of 12 homozygous for CYP2C19m1 (m1/m1), three homozygous for CYP2C19m2 (m2/m2), and 20 heterozygous for the two defects (m1/m2). Thus the allele frequencies of CYP2C19m1 and CYP2C19m2 were calculated to be 0.29 and 0.13 (107 and 46 of the total of 372 alleles), respectively. Among the 46 subjects phenotyped, seven were identified as the poor metabolizers, with a log10 urinary excretion of 4'-hydroxymephenytoin of < 0.3% of the racemic dose. These seven subjects were genotyped as the individuals with the m1/m1 (two), m1/m2 (four) or m2/m2 (one) allele combination, indicating a complete concordance between the phenotyping and genotyping tests., Conclusion: The present genotyping test confirmed that the frequency of CYP2C19 mutant gene m1 is about 2.2 times greater than another mutant gene, m2, among Japanese poor metabolizers. The genotyping of CYP2C19 discriminates between the two S-mephenytoin 4'-hydroxylation phenotypes completely in the Japanese subjects.

The pharmacokinetic profile of omeprazole was examined in 27 healthy Japanese volunteers, and the results were analyzed in relation to genotype for the two mutations, CgammaP2C19m1 in exon 5 and CgammaP2C19m2 in exon 4, associated with the poor metabolizer phenotype. Of the 27 individuals analyzed, 10 were homozygous for the wild-type (wt) allele in both exon 5 and exon 4 (wt/wt; 37.0%, pattern GI), five were heterozygous for the CgammaP2C19m1 (wt/m1; 18.5%, G2), five were heterozygous for the CgammaP2C19m2 (wt/m2; 18.5%, G3), two were heterozygous for the two defects (m1/m2; 7.4%, G4), and five were homozygous for the CgammaP2C19m1 (m1/m1; 18.5%, G5). The allele frequencies of the m1 and m2 mutation were 0.31 and 0.13, respectively. A correlation between the rate of metabolism of omeprazole and genotype was observed. The mean clearance values of omeprazole in patterns G1, G2, G3, G4, and G5 were 1369.0, 332.7, 359.0, 70.8, and 89.5 ml/hr/kg, respectively. The relative area under the serum concentration-time curve (AUC) ratio of omeprazole to 5-hydroxyomeprazole in patterns G1, G2, G3, G4, and G5 was 1:2.8:3.4:16:17.2. A similar relation was observed in the omeprazole/5-hydroxyomeprazole serum concentration ratio, determined 3 hours after drug intake (1:3:4:18.8:20.3). There were significant (p < 0.05 to 0.01) differences in the disposition kinetics of omeprazole between the subjects with patterns G1, G2, and G3 and the subjects with patterns G4 and G5. The results indicate that the 5-hydroxylation pathway of omeprazole is clearly impaired in subjects with m1/m2 and m1/m1.

1. Interethnic differences in cytochrome P4502A6 (CYP2A6) levels and coumarin 7-hydroxylation activities were determined in liver microsomes of 30 Japanese and 30 Caucasians. 2. Although CYP2A6 levels and coumarin 7-hydroxylation activities varied very significantly in the 60 human samples examined, both CYP2A6 levels and coumarin 7hydroxylation activities were found to be higher in Caucasian than Japanese population. 3. Interestingly, eight of the 30 Japanese examined showed very low or undetectable levels of coumarin 7-hydroxylation activities as well as of CYP2A6 in liver microsomes. All of the Caucasians, however, had significant CYP2A6 levels and variable 7-hydroxylation activities. 4. Kinetic analvsis of coumarin 7-hydroxylation activities in liver microsomes of various human samples suggested that although there were 260-fold differences in Vmax's in 10 human samples examined, the Km's were very similar (2.1 + or - 107 mu M); a value consistent with that obtained (1.2 mu M) with purified CYP2A6 in reconstituted system. 5. The results suggest that CYP2A6 is actually involved in the 7-hydroxylation of coumarin in human liver microsomes, and that interethnic differences in coumarin 7-hydroxylation activities in Japanese and Caucasian population may be ascribed to the differences in expression of CYP2A6 protein.

It has recently been shown that the most common mutation (named m1) in both Caucasian and Japanese poor metabolizers (PM) of S-mephenytoin is a single base pair mutation (G-->A) in exon 5 of the CYP2C19 gene. In Japanese, a second defective allele of CYP2C19 named m2 consists of a G-->A mutation in exon 4. In the present study, we have investigated the inheritance of the CYP2C19 wild type allele (wt) and the two defective alleles (m1 and m2) in families of 11 Danish PM probands. The study was carried out for two principal reasons. First, we wanted to confirm the autosomal recessive inheritance of the defective alleles, and second, we wanted to examine the specificity and sensitivity of the CYP2C19 genotyping test. Individuals were phenotyped by measuring the ratio of S/R mephenytoin excreted in the urine after administration of mephenytoin, and genotyping was carried out by a PCR-based DNA amplification procedure. The genotypes of nine of the 11 probands were consistent with their phenotypes. Eight were homozygous m1/m1, and one was heterozygous m1/m2. The genotypes of two putative PM probands (wt/m1) were not consistent with their phenotypes. On the basis of extended phenotyping (additional late urine collections (24-36 h) and acidification of urine), one of these could probably be reclassified as an extensive metabolizer (EM) while the other was considered to be a true PM. This suggests the presence of an additional unknown mutant allele in the latter. Seven of the 41 phenotyped relatives in the 11 families were phenotyped as PMs, and with the exception of the father of family 10, their genotypes (m1/m1) were consistent with their phenotypes. Extended phenotyping (acidification of urine) suggested that the father of family 10 in fact is an EM and hence that his genotype (wt/m1) is concordant with his phenotype. Thus, the specificity of genotyping tests for PM was 100%, while the sensitivity was 15/16 or 94%. Our study provides unequivocal evidence for autosomal recessive inheritance of the PM trait.

A genetic polymorphism in the metabolism of the anticonvulsant drug (S)-mephenytoin has been well documented in humans. There are marked interracial differences in the frequency of the poor metabolizer phenotype, which comprises 2-5% of Caucasian but 18-23% of Asian populations. We have recently reported that the principal genetic defect responsible for the poor metabolizer phenotype is a single-base pair mutation in exon 5 of CYP2C19 (CYP2C19m), which accounts for approximately 75-83% of the defective alleles in both Japanese and Caucasians subjects. In the present study, we have identified a new mutation (CYP2C19m2) in Japanese poor metabolizers, consisting of a guanine to adenine mutation at position 636 of exon 4 of CYP2C19, which creates a premature stop codon. Genotyping of seven Japanese poor metabolizers who were not homozygous for the previously described CYP2C19m defect (now designated CYP2C19m1) indicated that they were either homozygous for the new defect (CYP2C19m2/CYP2C19m2) or heterozygous (CYP2C19m1/CYP2C19m2) for the two defects. CYP2C19m1 accounts for 25 of 34 alleles in Japanese poor metabolizers, whereas CYP2C19m2 accounts for the remaining nine alleles. Hence, CYP2C19m1 and CYP2C19m2 explain 100% of the available Japanese poor metabolizers (34 alleles). In contrast, the CYP2C19m2 defect was not detected in nine Caucasian poor metabolizers (83% of available poor metabolizer alleles were CYP2C19m1), indicating the existence of another, as yet unidentified, mutation. Genetic testing of the families of two Japanese poor metabolizer probands showed that coinheritance of the CYP2C19m1 and CYP2C19m2 alleles was concordant with the autosomal recessive inheritance of the poor metabolizer phenotype.